Differential Expression of Uridine Phosphorylase in Tumors Contributes to an Improved Fluoropyrimidine Therapeutic Activity

Published OnlineFirst September 27, 2011; DOI: 10.1158/1535-7163.MCT-11-0202

Molecular Cancer Preclinical Development Therapeutics

Deliang Cao1, Amy Ziemba4, James McCabe3, Ruilan Yan1, Laxiang Wan2, Bradford Kim2, Michael Gach4, Stuart Flynn3, and Giuseppe Pizzorno2,4

Abstract Abrogation of uridine phosphorylase (UPase) leads to abnormalities in pyrimidine metabolism and host protection against 5-fluorouracil (5-FU) toxicity. We elucidated the effects on the metabolism and antitumor efficacy of 5-FU and capecitabine (N4-pentyloxycarbonyl-50-deoxy-5-fluorocytidine) in our UPase knockout (UPase / ) model. Treatment with 5-FU (85 mg/kg) or capecitabine (1,000 mg/kg) five days a week for four weeks caused severe toxicity and structural damage to the intestines of wild-type (WT) mice, but not in UPase / animals. Capecitabine treatment resulted in a 70% decrease in blood cell counts of WT animals, with only a marginal effect in UPase / mice. UPase expressing colon 38 tumors implanted in UPase / mice revealed an improved therapeutic efficacy when treated with 5-FU and capecitabine because of the higher maximum tolerated dose for fluoropyrimidines achievable in UPase / mice. 19F-MRS evaluation of capecitabine metabolism in tumors revealed similar activation of the prodrug in UPase / mice compared with WT. In WT mice, approximately 60% of capecitabine was transformed over three hours into its active metabolites, whereas 80% was transformed in tumors implanted in UPase / mice. In UPase / mice, prolonged retention of 0 5 dFUR allowed a proportional increase in tumor tissue. The similar presence of fluorinated catabolic species confirms that dihydropyrimidine dehydrogenase activity was not altered in UPase / mice. Overall, these results indicate the importance of UPase in the activation of fluoropyrimidines, the effect of uridine in protecting normal tissues, and the role for tumor-specific modulation of the phosphorolytic activity in 5-FU or capecitabine-based chemotherapy. Mol Cancer Ther; 10(12); 2330–9. 2011 AACR.

Introduction (50-dFUR), and capecitabine (N4-pentyloxycarbonyl-50- deoxy-5-fluorocytidine). UPase knockout (UPase / ) mice Uridine phosphorylase (UPase), a phosphorolytic en- provide an ideal model for this study (6). zyme ubiquitously expressed, has been shown to be The antitumor activity of 5-FU stems from its proximal 0 0 induced in various human solid tumors compared with metabolites, 5-fluoro-2 -deoxyuridine-5 -monophosphate 0 surrounding normal tissues (1–2). This is possibly due to (FdUMP), 5-fluorouridine-5 -triphosphate (FUTP), and 0 0 frequent mutations of p53 (a suppressor of UPase gene 5-fluoro-2 -deoxyuridine-5 -triphosphate (FdUTP). These expression; ref. 3) or higher expression of various cyto- active metabolites either inhibit the activity of thymidy- kines (inducers of UPase expression) in tumor tissues late synthase or incorporate into RNA or DNA, leading to (4, 5). Because of its increased expression in tumors, nucleic acid dysfunction and cell death (7, 8). Several it is important to determine the role of UPase in the studies have indicated the role of UPase in fluoropyrimi- activation and antitumor activity of fluoropyrimidines, dine activation, including the anabolism of 5-FU into 5- such as 5-fluorouracil (5-FU), 50-deoxy-5-fluorouridine fluorouridine (FUrd) with subsequent phosphorylation to 5-fluorouridine monophosphate (FUMP) and the phosphorolysis of the prodrug 50-dFUR into 5-FU (9–11). Using fi 1 Authors' Af liations: Department of Medical Microbiology, Immunology, human and murine cancer cell lines, Peters and colleagues and Cell Biology, Simmons/Cooper Cancer Institute, Southern Illinois University School of Medicine, Springfield, Illinois; Departments of 2Internal (12) reported that FUrd synthesis was directly correlated Medicine (Medical Oncology) and 3Pathology, Yale University School of to the intracellular UPase activity. In colon 26 tumor Medicine, New Haven, Connecticut; and 4Division of Drug Development, Nevada Cancer Institute, Las Vegas, Nevada cells, a mixture of TNF-a, interleukin-1a (IL-1a), and IFN-g efficiently enhanced 5-FU cytotoxicity 2.7-fold Corresponding Author: Giuseppe Pizzorno, Division of Drug Develop- 0 ment, Nevada Cancer Institute, One Breakthrough Way, Las Vegas, NV and 5 -dFUR cytotoxicity 12.4-fold due to induction of 89135. Phone: 702-822-5380; Fax: 702-944-2369; E-mail: UPase activity (5). However, in most experimental models [email protected] the contribution of UPase to fluoropyrimidine activity has doi: 10.1158/1535-7163.MCT-11-0202 been controversial due to the coexistence of other related 2011 American Association for Cancer Research. metabolic enzymes, thymidine phosphorylase (TPase)

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Uridine Phosphorylase and Fluoropyrimidine Activity

and orotate phosphoribosyl transferase (OPRTase). OPR- guidelines for the humane treatment of animals. Colon 38 Tase participates in the de novo pyrimidine synthe- murine adenocarcinoma cells (MC38) were originally sis pathway, directly converting 5-FU to FUMP in the obtained from the Southern Research Institute, Birming- presence of 5-phosphoribosyl-1-pyrophosphate (PRPP; ham, AL (19) and authenticated by flow cytometry before ref. 13), whereas TPase contributes to the formation of implantation. fluorodeoxyuridine (dFUrd; ref. 14). In addition, TPase is also involved in the phosphorolysis of 50-dFUR into 5-FU In vivo toxicity of capecitabine (15). We have investigated the effect of UPase on the Toxicity of capecitabine was evaluated in WT and antiproliferative activity of fluoropyrimidines using a UPase / mice at 8 to 12 weeks. Mice were randomly gene-targeted cell model, UPase gene-knockout murine grouped according to gender and body weight, 6 mice embryonic stem cells, and found that the abrogation of per group. Capecitabine (Xeloda) was suspended in 40 UPase in these cells resulted in an 8- and 16-fold increase mmol/L citrate buffer (pH 6.0)/5% wt/vol of hydroxy- 0 in the IC50 values of 5-FU and 5 -dFUR, respectively (9). propylmethylcellulose and administered by oral gavage The unique genetic modification of UPase activity directly at 1,000 to 1,375 mg/kg daily, 5 days a week for 4 weeks. confirmed the role of UPase in fluoropyrimidine metab- Animal weights were measured daily to monitor the olism and activation. However, this in vitro system does toxicity. The dose leading to 15% to 20% weight loss was not allow the investigation of the metabolic kinetics and defined as the maximum-tolerated dose (MTD). Observa- tissue distributions of fluoropyrimidines. In addition, the tions of a given treatment group ceased when animals lost cultured cells are also limited for the study of capecita- more than 20% of their body weight, or after the first bine, a prodrug of 5-FU, because its activation is preclud- mouse death occurred in the group. All the experiments ed by the lack of carboxylesterase (16). In this article, we were conducted at least in duplicate. investigated the metabolic kinetics and tissue distributions of 5-FU in UPase / mice and evaluated the histo- Antitumor activity of 5-FU and capecitabine logic basis of host toxicity of capecitabine. To observe the therapeutic efficacy of fluoropyrimidine A major drawback of fluoropyrimidine-based chemo- treatment, murine colon 38 tumor suspension (200 mL) therapy are the severe dose-limiting side effects at the was implanted into both flanks of the mice (19). Colon 38 level of the bone marrow and gastrointestinal tract, often expresses WT UPase and its enzymatic activity was found resulting in therapeutic failure (17, 18). Therefore, the to be similar once transplanted in WT or UPase / mice development of prodrugs or modulatory strategies to (Table 1). When the tumors grew to an average size of increase tumor selectivity is an important effort to 100 to 150 mg, animals were randomly assigned into improve the therapeutic efficacy of fluoropyrimidines. groups, 6 mice in each group with comparable average Using UPase / mouse-based colon tumor models, we body weight and tumor size. Tumor size was determined assessed the effect of the tumor-specific modulation of by measuring the 2 axes of the tumor (L, longest axis, and UPase activity on tumor selectivity and antitumor efficacy W, shortest axis) with a Vernier caliper and weight esti- of 5-FU and capecitabine. The results provide important mated according to the following formula: Tumor-weight information for clinical approaches to improve the ther- (mg) ¼ W2 L/2 (19). 5-FU was administered weekly apeutic outcomes of fluoropyrimidine-based regimens. by intraperitoneal injection at 85 and 150 mg/kg, and capecitabine was administered orally on a daily basis at Materials and Methods 1,000 and 1,375 mg/kg, 5 days a week.

Animals and cell lines Metabolism and tissue distribution of 5-FU Wild-type (WT) and UPase / mice were produced and [6-3H]-5-FU (20 Ci/mmol, 200 mg/kg) was intrave- maintained as previously described (6). All experiments nously administered to mice through the tail vein. At the were carried out according to Yale University and NVCI indicated time points, animals were anesthetized and

Table 1. Enzymatic activities in tissues of WT and UPase/ mice (nmol/mg/h)

UPase TPase UK OPRTase DPD

WT UPase/ WT UPase/ WT UPase/ WT UPase/ WT UPase/

Liver 5.6 1.5 1.3 0.3 25.9 6.2 26.7 7.3 0.9 0.3 1.6 0.3a 2.3 0.4 2.1 0.5 2.0 0.3 2.0 0.4 Small intestine 696.8 80.0 ND 1.9 0.5 2.0 0.5 0.4 0.2 0.5 0.2 0.8 0.3 0.7 0.4 —— Colon 38 28.5 4.7 27.9 5.3 0.2 0.1 0.2 0.1 6.6 1.5 6.8 1.3 2.5 0.2 2.5 0.3 ——

Abbreviations: TPase, thymidine phosphorylase; UK, uridine kinase; OPRTase, orotate phosphoribosyltransferase. þ/þ, UPase WT; /, UPase knockout; ND, not detectable. a, P < 0.05 compared with WT mice.

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blood collected by retro-orbital bleeding. Plasma was ing 200 mmol/L [14C]-5-FU, 1 mmol/L PRPP, 100 mmol/L separated by centrifugation at 2,000 g,4 C for 5 minutes, MgCl2, 50 mmol/L Tris-Cl (pH 7.6), and 100 mmol/L BAU extracted with 2 volumes of 15% trichloroacetic acid was incubated at 37C for 1 hour. Uridine kinase was (TCA) at 14,000 rpm for 10 minutes and neutralized with detected by measuring the conversion of uridine into an equal volume of trioctylamine-freon (45:55, v:v). Aque- UMP in the presence of ATP (6). The reaction was done ous phase was used for high-performance liquid chroma- in a 100 mL volume with 200 mmol/L [14C]-uridine, 1 tography (HPLC) analysis (19). Tissues frozen in liquid mmol/L ATP, 100 mmol/L MgCl2, 50 mmol/L Tris-Cl nitrogen were weighed and homogenized in 2 volumes of (pH 7.6), and 100 mmol/L BAU. The resultant [14C]-UMP 15% TCA and the supernatant extracted for HPLC anal- was separated on TLC plates and determined by scintil- ysis as described for plasma. 5-FUrd and 5-FU were lation counting. Dihydropyrimidine dehydrogenase separated on a C18 reverse-phase Microsorb column (DPD) activity was measured by determining the con- (250 4.6 mm, Varian) using mobile phase ddH2O:10 version of uracil into dihydrouracil. The assay mixture mmol/L H3PO4:30 mmol/L heptane sulfonic acid contained 200 mmol/L NADPH and 8.25 mmol/L (94:5:1, pH 3.1) at 1.0 ml/min by cooling the column at [14C]-uracil in a solution constituted by 35 mmol/L 8C. 5-Fluororibonucleotides (FUXP), measured as FUMP potassium phosphate (pH 7.4), 2.5 mmol/L magnesium obtained after boiling the samples at 100C for 10 minutes, chloride, 10 mmol/L 2-mercaptoethanol. The formed were separated using a Whatman Partisil-10-SAX anion- [14C]-dihydrouracil was separated on TLC plates and exchange column with 5 mmol/L sodium phosphate radioactivity determined (22). mobile phase (pH 3.3) at 1.4 ml/min. Elution was frac- tioned and radioactivity determined using a scintillation MRS evaluation of capecitabine metabolism counter (Beckman). For RNA incorporation of 5-FU, liver, WT or UPase / mice bearing colon 38 tumors were tumor, and intestinal tissues were weighed and total RNA treated orally with 1,000 mg/kg capecitabine. Animals extracted using TRIzol reagent (Invitrogen; ref. 9). DNA were anesthetized with isoflurane (2%–3% induction and was isolated by suspending the precipitated nucleic acids 1%–2% maintenance) along with 0.5 LPM O2. MRI and in lysis buffer, then incubating the suspension at 55C MRS were done using a Bruker 7T/20 Biospin MRI, overnight and subsequently for 1 hour at 37C with RNase equipped with a dual-tuned 7 cm ID 1H/19F volume coil A/T1. To precipitate DNA, an equal volume of 2-propa- (Bruker), and a 15-mm ID detunable receive-only 19F nol was added and mixed until precipitation was com- surface coil (Doty Scientific). The surface coil was placed plete and further by extractions with phenol/chloroform/ over the region of interest (ROI) and the animal secured to 1 iso-amylalcohol (50/49/1 v/v/v) and chloroform/iso- minimize the effects of motion on the MR data. A HT2- amylalcohol (49/1 v/v). DNA was precipitated with an weighted TurboRare sequence [repeat time (TR): 2,330 equal volume of 2-propanol and reconstituted in 0.5 mL milliseconds, echo time (TE): 36 milliseconds, Rare factor: digestion buffer at 55C. For measurement of concentra- 8, matrix: 256 256, field of view: 4 cm] was used for tion and purity, optical density was measured at 260 and positioning and structural imaging of the animal and 280 nm. The radioactivity in DNA/RNA was determined delineating the ROI. A 1H point-resolved spectroscopy by scintillation counter (20). sequence was used for magnetic field shimming over the ROI (TR: 721 milliseconds, TE: 20 milliseconds, flip angle: Analysis of FdUMP concentration in tumors 90 degree; ref. 23). Colon 38 tumors were excised 4 hours after adminis- 19F spectra were acquired and averaged during 27.5 tration of 200 mg/kg of 5-FU and immediately frozen in minutes blocks [3,300 free induction decays (FID), TR: 0.5 liquid nitrogen. Frozen tumors were pulverized, sus- seconds, spectral width: 60 kHz, points/FID: 890, flip pended in 3 volumes of Tris-HCl buffer (pH 7.4), and angle: 60 degree, RF pulse duration: 50 mseconds] for 3 extracted with TCA (final concentration 5%). The super- hours. The 19F MRS data were analyzed using Bruker’s natant was neutralized and stored until analysis for Topspin application.Therate ofcapecitabineactivation and FdUMP. FdUMP was measured using a dilution assay buildup of the intermediate molecules were evaluated. based on the capacity of FdUMP present in tumors to inhibit the binding of [6-3H]-FdUMP to Lactobacillus casei Peripheral blood cells counting thymidylate synthase (21). Peripheral blood samples were collected from mice treated with capecitabine at 1,000 mg/kg for 4 weeks. Enzyme activity assays Number of erythrocytes per microliter and hematocrit UPase activity was measured by conversion of uridine values were determined using a Coulter counter (Model to uracil as described previously (9). TPase activity was ZF, Coulter Electronics). Hemoglobin was measured by a determined by measuring the conversion of thymidine hemoglobinometer (Coulter Electronics) using the cyan- into thymine using [3H]-thymidine as substrate and 100 methemoglobin method. Numbers and subtypes of white mmol/L 5-benzylacyclouridine (BAU, a specific UPase blood cells were determined on 50 mL of peripheral blood, inhibitor). OPRTase activity was assayed by measuring after red blood cells were osmotically lysed, using a flow the formation of 5-FUrd monophosphate from 5-FU in the cytometer (Becton Dickinson). Total and differential white presence of PRPP (9). A 100 mL reaction mixture contain- blood cell counts were determined. To determine the

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platelet counts, the white blood cell preparations were degradation pathway (25). When the drug is administered diluted at 1:10 and the counts were obtained by appro- orally or intraperitoneally, liver represents the major site priately adjusting gains and threshold (24). of 5-FU metabolism, with more than 85% of a given dose of the fluoropyrimidine eliminated through the rapid Histologic examination of small and large intestine formation of dihydrofluorouracil (26). Although the ex- The intestinal tract, the small intestine, and colon were pression and the enzymatic activity of DPD in the liver excised from mice treated with 5-FU (85 mg/kg) or cape- of both mice are virtually similar (Table 1), the elevated citabine (1,000 mg/kg) for 4 weeks. The isolated tissues 5-FU clearance in UPase / mice possibly suggests a re- were then fixed in formal fixative and transverse sections duced competition of uracil for DPD, in which uracil (5 mm) were prepared for staining with hematoxylin and formation from uridine degradation is blocked because eosin. Histologic changes of the tissues were evaluated of the lack of UPase. However, the plasma pharmaco- under light microscopy. kinetics of FUrd, following 5-FU delivery, displayed a significant difference in UPase / mice (Table 2). In WT Results mice, FUrd appeared in plasma within 5 minutes following intraperitoneal administration of 5-FU (200 mg/kg), In a previous study, we reported that the abrogation of reaching nearly 4 mmol/L at 10 minutes. Thereafter, FUrd UPase in mice led to a decrease in 5-FU host toxicity (6). concentration quickly declined to an undetectable level However, it was not clear whether this host protection at 4 hours. Plasma FUrd in UPase / mice was barely resulted from a reduced activation of 5-FU through the detectable at 10 minutes, and its peak appeared 1 hour pyrimidine salvage pathway or from the protection of after administration of the same dose of 5-FU. The clear- increased uridine in the plasma and tissues, due to ance of plasma FUrd was significantly slower in UPase / the elimination of UPase activity. In this study on the mice than in WT, and a considerable amount of FUrd was UPase / mouse model, we investigated metabolism and still detected 4 hours after 5-FU administration resulting tissue distribution of 5-FU and examined the pathologic in a significantly higher AUC (316 vs. 105 mmol/L-min; basis of 5-FU and capecitabine toxicity, and we evaluated Table 2).In WT mice, UPaserapidly catalyzed the formation the effect of UPase abrogation on the antitumor activity of of FUrd from administered 5-FU. However, in UPase / 5-FU and capecitabine. mice, plasma FUrd was possibly originated from the deg- Plasma clearance of 5-FU displayed a faster rate in radation of FUMP synthesized by OPRTase-catalyzed / UPase mice than in WT with a t1/2 of 26.3 minutes versus de novo pathway (13, 27) and eventually cleared by kidney 37.9 minutes and clearance of 29.4 versus 18.1 ml/min/kg, excretion (6). As a result, a delay occurred in plasma respectively, for UPase / and WT C57 BL6 mice (Table 2). peak concentration of FUrd, accompanied with a prolonged This indicates that alterations in the anabolic pathway of half-life in UPase / mice. 5-FU due to UPase abrogation significantly affected its The liver is the major metabolic organ for 5-FU, and the clearance rate, consequently reducing exposure to the gastrointestinal system represents one of the major tox- drug as indicated by a significantly smaller systemic expo- icity targets for fluoropyrimidines. Therefore, the 5-FU sure with an AUC of 84.5 mmol/L-min in WT compared metabolic pharmacokinetics and distribution in these with 52.3 mmol/L-min for the knockout mice. Normally, tissues were examined. Overall, 5-FU and FUrd showed plasma 5-FU is mainly removed via the DPD-initiated similar metabolic patterns in these tissues unlike that in

Table 2. Plasma pharmacokinetic parameters of 5-FU and derived 5-FUrd in WT and UPase/ mice following a bolus administration of 200 mg/kg of 5-FU

5-FU 5-FUrd

Parameter Units WT UPase/ WT UPase/

Tmax min 5 5 10 60

Cmax mmol/L 3,175.4 2,500.7 3.55 2.71

C0 mmol/L 5,333.1 5,074.6 ——

AUCinf mmol/L-min 85.177 52.385 0.104 —

AUC0–240 mmol/L-min 84.523 52.338 0.105 0.316

T1/2 min 37.9 26.3 —— CL mL/min/kg 18.1 29.4 ——

Vss mL/kg 633 595.7 —— NOTE: Estimated using WinNonlin V5.2, noncompartmental IV bolus model. Linear up, log down trapezoidal integration rule.

AUCinf value is predicted from terminal elimination rate constant. Treated the 0 concentration at 240 min for FURD WT as missing.

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AB 5-FU 5-FUrd 1,500 20 Liver, WT Liver, WT 1,250 Liver, KO Liver, KO Int, WT 15 Int, WT 1,000 Int, KO Int, KO

750 10 Figure 1. 5-FU metabolism and incorporation into liver and intestine 500 RNA. [3H]-5-FU was intravenously 5 administered at 200 mg/kg. The 250 5-FU Concentration (nmol/g) 5-FU Concentration (nmol/g) FUrd Concentration mouse liver and intestinal tissues 0 0 were collected at 10, 30, 60, and 0 60 120 180 240 300 0 60 120 180 240 300 240 minutes after injection. Time (min) Time (min) Amounts of 5-FU (A), FUrd (B), FUXP (C), and incorporation of CD 5-FU into RNA (D) were measured FUXP nucleotides formation 5-FU incorporation into RNA as described in Materials and 40 100 Methods. Values represent results of tissues pooled from 3 mice. KO, 35 Liver, WT 90 Liver, KO 80 UPase knockout; FUXP, 30 Int, WT ﬂuorouridine phosphates; Int, 70 Liver, WT Int, KO intestine. 25 60 Liver, KO Int, WT 20 50 Int, KO 15 40 30 10 20

5 RNA Incorporation (nmol/g) FUXP Concentration (nmol/g) FUXP Concentration 10 0 0 0 60 120 180 240 300 0 60 120 180 240 300 Time (min) Time (min)

plasma (Fig. 1A and B). 5-FU was cleared slightly faster in FUXP levels and 5-fluoro-RNA amounts in liver, gastro- both liver and intestines of UPase / mice, but with no intestinal tract and transplanted colon 38 tumor. As statistical significance (P > 0.05) compared with WT ani- shown in Fig. 1C and D, the levels of FUXP and 5- mals; although the formation and metabolism of FUrd fluoro-RNA were significantly higher in WT liver and varied with UPase expression status and tissue types. In intestines than in UPase / . 5-FU incorporated into RNA WT intestinal tissues, a high level of FUrd was detected at of liver tissue started to be cleared within the first hour, 10 minutes after intraperitoneal administration of 5-FU although we observed a protracted accumulation in the (200 mg/kg) but declined rapidly within 30 minutes RNA of the intestinal tissues of WT mice for more than 4 and still maintained detectable levels after 4 hours. In hours, providing the pharmacokinetic basis for the intes- UPase / intestinal tissues, however, FUrd appeared at 30 tinal lesions caused by the fluoropyrimidines in the WT minutes and peaked at 60 minutes after 5-FU delivery. mice. The lower incorporation of 5-FU in the normal However, in UPase / liver tissues, FUrd was present at 10 tissues of the UPase / mice, likely due to the competition minutes and reached a peak concentration at 30 minutes, of high uridine levels, confirms our previous observation 3-fold higher than that in the intestines, indicating an of reduced in vivo host toxicity to 5-FU and a 75% higher active 5-FU anabolism through pyrimidine de novo syn- MTD of 5-FU in this strain (6). thesis in liver. Similar to that in the intestines, the clear- We observed significant differences in the concentra- ance of FUrd was much faster in WT liver than that in tion of uridine in plasma and normal tissues of WT mice UPase / (Fig. 1B). Taken together, these data indicate the compared with the UPase / (Table 3). Uridine concen- important role of UPase in 5-FU/FUrd metabolism in tration was elevated 3-fold in the intestine and kidney and normal intestinal and liver tissues, supporting our previ- up to 15-fold in spleen of UPase / mice compared with ous observation that ribose-1-phosphate is not a rate- the corresponding WT tissues. In colon 38 tumors trans- limiting factor in the 5-FU anabolic metabolism catalyzed planted in both WT and UPase / mice, we observed a by UPase (9). reduced uridine concentration compared with the other The incorporation of 5-FU into RNA is important for its tissues with levels similar to plasma concentrations. The antiproliferative activity (28) but has also been linked to elevation in tumor uridine concentration we determined the toxic effect of fluoropyrimidines (29–30). Therefore, in UPase / was highly significant (P < 0.01) compared we included in our investigation the measurement of with the tumor in WT mice (7 vs. 1 mmol/L); however, the

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Table 3. Plasma and tissue concentrations of endogenous uridine in WT and UPase/ mice

Plasma Tissues (pmol/mg) (mmol/L)

sd Gut sd Kidney sd Liver sd Spleen sd Tumor sd

WT 1.5 0.6 29.3 3.4 24. 6 3.6 6.4 2.5 4.9 1.1 1.1 0.2 UPase/ 7.2 2.5 89.3 2.3 71.0 12.6 42.8 7.0 75.2 12.8 7.6 0.7 uridine concentration still remained almost 10-fold below blood cell counts were used to evaluate the hematologic the normal tissues concentrations (Table 3). toxicity, and intestinal transverse sections were examined When we measured the incorporation of 5-FU in colon following hematoxylin and eosin staining to evaluate the 38 tumors, we observed 69.2 28.4 nmol/g into the RNA intestinal lesions. The treatment with capecitabine (1,000 of tumors in WT mice and 64.6 26.9 nmol/g in tumors mg/kg) for 4 weeks caused a decrease of up to 70% in implanted in UPase / 4 hours after the administration of peripheral blood cells of WT animals. The alterations a 200 mg/kg dose of 5-FU. Also the incorporation of 5-FU occurred in the hematocrit level (40%), red blood cells into DNA was not significantly different between tumors (44%), white blood cells (72%) and subtypes, and grown in WT mice compared with UPase / , 367 57 platelets (66%), indicating an overall effect on the regen- fmol/mg of DNA and 431 83 fmol/mg of DNA, respec- eration and differentiation of bone marrow cells. These tively. The concentration of FdUMP present in colon 38 decreases in cell counts were minimal and not significant tumors following a 200 mg/kg administration of 5-FU did in the peripheral blood of UPase / mice, indicating that not show any significant difference between the tumors the abrogation of UPase activity and the high concentra- implanted in the 2 different strains, 66 21 pmol/g of tion of circulating uridine protected the bone marrow tissue of FdUMP in WT versus 72 13 in UPase / mice. from capecitabine-induced toxicity. A similar result was The role of UPase in the activation of 50-dFUR to 5-FU also observed in mice treated with 5-FU (data not shown). has also been determined in this study. Capecitabine is a The UPase abrogation also protected murine intestine prodrug of 5-FU, approved for the treatment of advanced from capecitabine lesions. We observed a complete de- breast and colon cancers (31, 32). The carbamate modifi- struction of the villous architecture, crypts, and muscle cation facilitates its passage through the gastrointestinal layer in the intestines of WT mice treated with 1,000 mg/kg mucosa without activation, leading to almost 100% oral of capecitabine for 4 weeks, but not in the intestines of bioavailability (33). In liver, capecitabine is hydrolyzed by UPase / mice exposed to the same treatments. This carboxylesterase to 50-deoxy-5-fluorocytidine (50-dFCR) protection by UPase abrogation confirms the critical role then converted to 50-dFUR by cytidine deaminase. 50- of UPase in 50-dFUR/capecitabine activation and toxicity. dFUR has no inherent cytotoxic activity; to exert its anti- To evaluate the clinical significance of host protection proliferative activity, it must be hydrolyzed to 5-FU by provided by UPase abrogation, we further assessed the UPase and TPase (17, 33). In this study, we assessed the antitumor efficacy of 5-FU and capecitabine (Fig. 2A). effect of UPase on the host toxicity of capecitabine using Treatments were initiated when the implanted tumors UPase / mice. A daily1,000 mg/kg oral dose of capeci- grew to an average size of 100 to 150 mg. 5-FU was used at tabine caused nearly 20% weight loss in WT mice within 4 85 mg/kg (the MTD for WT mice) and 150 mg/kg (a dose weeks; and 1,375 mg/kg led to the first death immediately shown to be tolerated only by UPase / mice; ref. 7); and preceding the third dose due to gastrointestinal bleeding capecitabine was administered at 1,000 mg/kg (the MTD (data not shown). In UPase / mice, however, no signif- for WT mice) and 1,375 mg/kg (a dose shown to icant host toxicity was observed at doses up to 1,250 be tolerated only by UPase / mice). As presented mg/kg, and the higher dose of 1,375 mg/kg caused weight in Fig. 2B, a higher dose of 150 mg/kg of 5-FU efficiently loss (15%) only comparable to that in WT mice at 1,000 controlled and reduced tumor size, whereas the standard mg/kg, further clarifying the role of UPase in the activa- 85 mg/kg dose of 5-FU only partially slowed down tumor tion of 50-dFUR/capecitabine. Possibly due to the signif- growth in both strains. In the capecitabine treatments, a icant TPase activity reported in mouse liver (6), which also 30% dose increase from 1,000 mg/kg to 1,300 mg/kg contributes to conversion of 50-dFUR into 5-FU (15, 34), a resulted in dramatic tumor growth inhibition in UPase / larger MTD dose was not achieved in the UPase / mice. mice (Fig. 2C), with complete disappearance of tumors As previously indicated, bone marrow and the gastro- in 10 of 11 mice. These data indicate that the tumor-specific intestinal tract are the 2 major targets of fluoropyrimidines modulation of UPase activity can greatly improve the lesions (17, 18). To understand the histologic basis of this antitumor efficacy of 5-FU and capecitabine by allowing host protection by UPase abrogation, we further examined dose escalation without causing significant host toxicity. the pathologic changes in the intestines and bone marrow To better evaluate the activation of capecitabine and the of animals treated with 5-FU and capecitabine. Peripheral potential differences in metabolism between colon 38

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A O

O CH3 HN O F F HN N

O N O N H H2 HO C O Figure 2. Effect of tumor-speciﬁc modulation of UPase on 5-FU and H HH capecitabine antitumor activity. A, H structural comparison of 5-FU and capecitabine. Colon 38 tumor OH OH models were established as 5-FU Capecitabine described in Materials and Methods. 5-FU (B) was BC intraperitoneally administered 5-FU antitumor activity Capecitabine antitumor activity weekly and capecitabine (C) was delivered orally, 5 days a week, at 1,400 1,400 the indicated doses. Body weight WT, 85 mg/kg WT, 1,000 mg/kg and tumor size were measured 1,200 WT, Saline Control 1,200 WT, Control ) KO, 85 mg/kg ) KO, 1,000 mg/kg every other day. Observations 3 3 KO, 1,300 mg/kg KO, 150 mg/kg ceased when 20% or more of body 1,000 1,000 KO, Control KO, Saline Control weight loss occurred. Presented 800 800 values represent an average SD of 9 to 11 tumors carried by 6 mice. 600 600

400 400 Tumor volume (mm volume Tumor Tumor volume (mm volume Tumor 200 200

0 0 0 5 10 15 20 25 0 5 10 15 20 25 Time (d) Time (d)

tumors implanted in WT mice and in UPase / mice, we 5.1 ppm, respectively, were impossible to quantitate, conducted a series of in vivo 19F-MRS experiments using a given the overwhelming presence of 50-dFCR and 50- 1,000 mg/kg oral dose of capecitabine administered as a dFUR in the 3.5 to 5 ppm region. bolus. The in vivo spectra (Fig. 3A) resolve 5 main fluo- Our data indicates that the genetically induced changes rinated species, with the administered drug capecitabine and the elevation in circulating uridine did not interfere at 7 ppm upfield from the 5-FU signal, 50-deoxy-5-fluor- with the carboxylesterases and cytidine deaminase in ocytidine at 5 ppm, 50-deoxy-5-fluorouridine at 3.5 ppm, the activation of capecitabine. Also, the comparable pres- and the 2 main fluorinated metabolites fluoroureidepro- ence of 5-FU catabolites FUPA and FbAL in liver from pionic acid and a-fluoro-b-alanine at 17 and 19 ppm, WT and UPase / mice confirms the similar DPD enzy- respectively. As reported in Fig. 3A, we detected all 5 matic activity (Table 1) and quantitative real-time RT-PCR fluorinated species mentioned, starting 30 minutes after data (WT 1.0 0.1 vs. 0.8 0.2 in UPase / ) and, therefore, the oral administration of capecitabine, and were able to a similar rate of catabolic degradation for 5-FU in the follow their presence over 3 hours of spectra acquisition. 2 murine strains. We observed in tumors implanted in WT mice that capecitabine represented approximately 35% of the total fluo- Discussion rine signal, 50-dFCR corresponded to a similar percentage, whereas 50-dFUR ranged from 25% to 30% of total fluo- UPase activity and expression has been shown to be rine. The 2 catabolites, FUPA and FbAL were approxi- elevated in many tumors including colorectal carcinomas, mately 5% of the total (Fig. 3B). The tumors implanted in breast cancer, melanomas, and lung adenocarcinomas the UPase / mice showed that capecitabine was approx- (11). Several mechanisms have been uncovered, all lead- imately 15% of the total fluorine signal, 50-dFCR 45%, 50- ing to an increased expression in human tumors. We have dFUR 35%, and the sum of the 2 catabolites ranged from previously reported that the expression of UPase is 4% to 7% of the total (Fig. 3C). induced by TNF-a through the NF-kB pathway (11). We were unable to capture the 5-FU signal consistently, Other groups have shown that EWS/ETS fusion proteins, likely due to the rapid transformation of 5-FU into 5-FUrd playing a dominant oncogenic role in cell transformation and its fluoronucleotides in colon 38 tumors expressing in Ewings family tumors, induce UPase gene expression WT UPase. 5-FUrd and FUXP with their signals at 3.6 and through interaction with the UPase promoter (35). More

2336 Mol Cancer Ther; 10(12) December 2011 Molecular Cancer Therapeutics

Uridine Phosphorylase and Fluoropyrimidine Activity

B Capecitabine 5′-dFCR 5′-dFUR Catabolites

A 40

30 90 min 5′-dFCR 20 10 % Fluorinated derivatives

5′-dFUR 0 30 60 90 120 150 180 Time (min)

Capecitabine C Capecitabine 5′-dFCR 5′-dFUR Catabolites 60

FUPA Fβal 50

% Fluorinated derivatives 0 30 60 90 120 150 180 Time (min)

Figure 3. Intratumoral metabolism of capecitabine by 19F-MRS. WT and UPase / mice bearing colon 38 tumors were treated orally with 1,000 mg/kg capecitabine. Animals were anesthetized with isoflurane and MRS was done using a Bruker 7T/20 Biospin MRI. A, presence of fluorinated species in colon 38 tumors implanted in UPase/ mice. B, distribution of capecitabine metabolites in colon 38 tumors implanted in WT mice. C, distribution of capecitabine metabolites in colon 38 tumors implanted in UPase/ mice over a 3-hour acquisition time. The values represent the averages of tumor measurements conducted in 6 to 8 mice per group SD. recently, PGC-1a/ERR–dependent upregulation of above 50 mmol/L with the gut, the major target of 5-FU UPase was shown to contribute to an increased enzymatic toxicity at 90 mmol/L. These concentrations have been activity in colon and breast cancer cells (36). These results found previously to be sufficient to provide adequate show that the elevation of UPase in tumor is a key protection against 5-FU–based chemotherapy regimens contributor to the tumor selectivity of 5-FU and capeci- (39–40). Similarly, the concentration of uridine in colon tabine. Unlike TPase, UPase is not associated with any 38 tumors implanted in UPase / mice approximated identified angiogenic activity because of its limited cata- the plasma uridine concentration of 7 mmol/L, indicat- lytic activity on deoxynucleosides. It has been shown that ing that the inability of some tumors to accumulate this 2-deoxyribose-1-phosphate released from the deoxynu- nucleoside is likely due to the loss of the concentrative cleoside by TPase activity can act as an endothelial cell transport mechanism (41). chemoattractant and angiogenic factor (37). This feature Several studies have shown the capacity of large doses allows for a convenient modulation of the phosphorolytic of uridine to reduce 5-FU toxicity, without affecting its activity in tumors by UPase gene transfer or delivery of antitumor activity, if properly sequenced (42). Unfortu- specific inducers of UPase gene expression, such as cyto- nately, the administration of large doses of uridine, kines. This modulation would not be complicated by because of its rapid half-life, results in moderate to severe possible tumor growth stimulation due to simultaneously toxicity. Our laboratory has shown that this problem induced angiogenesis, as is the case with TPase (38). could be overcome by using inhibitors of UPase, such as The data here presented establish once more the role BAU, to conserve endogenous uridine with consequent of uridine in protecting normal tissues from the toxicity elevation of its concentration in plasma and tissues. A of fluoropyrimidines. In the normal tissues of UPase / phase I clinical trial of oral BAU administered as a single mice, we observed a constitutive uridine concentration agent has shown the ability of this inhibitor to elevate the

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Cao et al.

plasma uridine concentration 2- to 3-fold without signif- studies have indicated that ENT1 is a sort of housekeeping icant host toxicity (43). transporter, and its expression is often high and is highly The combination "rescue regimens" of 5-FU plus uri- retained in tumors (48). dine were initially proposed to evaluate the hypothesis A clinical study using a tissue array on 300 paraffin- that the antitumor effect of 5-FU is primarily due to the embedded gynecologic tumors (endometrium, ovary, and inhibition of thymidylate synthase and the host toxicity cervix) showed that hENT1 and hENT2 protein expres- mostly caused by the incorporation of the fluoropyrimi- sion were highly retained, but a significant number of dine into RNA (44). In vivo studies in a murine model and tumors were hCNT1 negative (49). A more recent breast in vitro data (45, 46) have clearly indicated that the incor- cancer study has shown that the percentage of hCNT1- poration of 5-FU into RNA seems to be the major cause of positive cells correlates positively with a reduced long- gastrointestinal toxicity that uridine inhibited the incor- term survival in patients treated with cyclophospha- poration and avoided the cytotoxic effect, whereas mide/methotrexate/5-FU chemotherapy (50). It is thymidine did not prevent 5-FU toxicity. reasonable to speculate that high expression of hCNT1 Clinical studies of 5-FU in combination with methotrex- could be linked to high nucleotide salvage efficiency ate and PALA have shown that patients tolerated combi- interfering with 5-FU nucleoside metabolism and, ulti- nation therapy with delayed uridine up to a weekly dose of mately, with its antiproliferative activity. 750 mg/m2 of 5-FU, with 25% experiencing moderate In summary, this study determined the metabolism and mucositis (grade II). In previous clinical trials without tissue distribution of 5-FU in UPase / mice and proved uridine, 4 of 6 patients could not tolerate a 600 mg/m2 the role of UPase in the activation and antitumor activity dose of 5-FU because of mucositis, diarrhea, and a of 5-FU and capecitabine. This study also showed the decrease in performance status. Another study of high- pathologic basis for host protection by UPase abrogation dose 5-FU with doxorubicin, high-dose methotrexate and from 5-FU and capecitabine lesions, the ability of uridine leucovorin, and oral uridine administration allowed for to protect the normal tissues, and exhibited the effect of dose intensification of 5-FU and ensured rescue from 5- tumor-specific expression of UPase on the therapeutic FU–induced hematologic toxicity without adverse impact efficacy of these agents. on tumor response (47). As indicated in preclinical studies, properly delayed uridine rescue results in a faster clear- Disclosure of Potential Conflicts of Interest ance of 5-FU from RNA of bone marrow and enhancement of the rate of recovery of DNA synthesis (39). No potential conflicts of interest were disclosed. Nucleoside transporters are expressed in a variety of cells and tissues with different substrate specificity and Grant Support selectivity. Most cell types coexpress concentrative nucle- This work was supported in part by grants from the National Cancer oside transporters (CNT) and equilibrative nucleoside Institute CA67035 and CA102121 (G. Pizzorno), the Charlotte Geyer transporters (ENT) to maintain their nucleoside supply. Foundation (G. Pizzorno), and the United States Army Medical Research Uridine is mainly accumulated in cells of different origin Breast Cancer Research Program DAMD17-00-1-0508 (D. Cao). þ The costs of publication of this article were defrayed in part by the by the Na -dependent CNT1 (SLC28A1) but also by payment of page charges. This article must therefore be hereby marked CNT3 (SLC28A3) that has broader substrate specificity. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ENTs, particularly ENT1 (SLC29A1) and ENT2 (SLC29A2), mediate facilitated diffusion transport with Received June 15, 2011; revised September 2, 2011; accepted September broader selectivity but relatively low affinity. Recent 5, 2011; published OnlineFirst September 27, 2011.

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Differential Expression of Uridine Phosphorylase in Tumors Contributes to an Improved Fluoropyrimidine Therapeutic Activity

Deliang Cao, Amy Ziemba, James McCabe, et al.

Mol Cancer Ther 2011;10:2330-2339. Published OnlineFirst September 27, 2011.

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